New conjugated molecules comprising a peptide derived from the cd4 receptor coupled to a polyanionic polypeptide for the treatment of aids

ABSTRACT

This invention relates to a conjugated molecule comprising a peptide derived from the CD4 receptor coupled to an organic molecule by means of a linker as well as a process for its preparation. Said organic molecule comprises a 5 to 21 amino acid anionic polypeptide. Such a conjugated molecule can be used in antiviral treatment, namely in the treatment of AIDS.

This invention relates to a conjugated molecule comprising a peptidederived from the CD4 receptor and an organic molecule, such as apolyanionic polypeptide. Such a conjugated molecule can be used inantiviral treatment, namely in the treatment of AIDS. This inventionfurther relates to processes for the preparation of the conjugatedmolecule.

Triple therapies combining nucleoside (NRTI), non-nucleoside (NNRTI)and/or protease inhibitors (PI) result in a reduction in viral chargebeneath levels of detection in a large number of seropositive HIVpatients. These therapies target reverse transcription and proteolysisat the same time. Their efficacy has led to a substantial decrease inthe number of deaths resulting from HIV infection. Unfortunately, about80% of the patients show genotypes with antiviral resistance and, moreworryingly, 45.5% of viral populations are resistant to NRTI/PIcombinations while 26% are resistant to a combination of three anti-HIVclasses (Tamalet et al., AIDS. 2003 Nov. 7; 17(16):2383-8). Thisobservation is particularly disturbing since the adverse effects oflong-term triple therapy treatment (lipoatrophy, lipodystrophy,hypertriglyceridaemia, hypercholesterolaemia, neuropathy, etc.) found in70% of patients receiving the treatment result in poor compliance and“sudden” discontinuation of treatment which often leads to resistance.The development of less severe forms of treatment with fewer adverseeffects and without cross-resistance is therefore a priority despite thelarge number of currently available medications on the market. With thisin mind, it is essential to target HIV replication steps other thanreverse transcription and proteolysis.

Entry of a virus into a cell is a crucial step in the viral infectioncycle. This process is divided into two phases: first, the virusinteracts with specific host receptors at the cell surface, followed bypenetration of the viral genetic material into the target cell. WithHIV, the molecular partners involved in the mechanisms of adhesion andentry are well established. The gp120 viral envelope glycoproteinessentially determines the virus/cell interaction complex, by binding toa transmembrane glycoprotein of the host cell, CD4.

This interaction leads to a conformational change in gp120 which exposesa particular epitope, called CD4-induced (CD4i), thus creating a bindingsite for chemokine receptors (essentially CCR5 and CXCR4). CCR5 andCXCR4 therefore act as gp120 co-receptors at the cell surface. Thissecond interaction leads to re-organization of the gp120/gp41 proteincomplex and initiation of cell/virus membrane fusion.

The cellular tropism of the HIV virus is defined by the type ofco-receptor used. So-called X4 or <<T-tropic>> viruses tend to infectmore specifically cell lines expressing CXCR4 at their surface, such asthe T lymphocytes. So-called R5 or <<M-tropic>> viruses use co-receptorCCR5 and mainly infect macrophages and monocytes. The presence of R5 orX4 viruses is generally associated with quite distinct stages of AIDSdevelopment (asymptomatic phase for R5, appearance of X4 virus oftenlinked to unfavourable evolution outcome of the disease, suggesting thatuse of co-receptor CXCR4 is an important factor in the pathogenesis ofAIDS). As the structural determinants for recognition of CCR5 and CXCR4are carried by gp120, the R5 and X4 viruses represent two separatetargets.

It was shown in international patent application WO 2008/015273 thatparticular activated peptides derived from the CD4 receptor can reactselectively with organic molecules affording chemically defined covalentconjugates. This activation requires the insertion of one and only oneresidue of the amino acid lysine in a defined position in the sequenceof the peptide derived from the CD4 receptor. This insertion allows thecoupling of organic compounds through linkers and chemistries after theminiCD4 synthesis and purification.

Numerous potential antiviral derivatives, consisting of conjugatedmolecules comprising a CD4 peptide specifically coupled to polyanionicheparan sulphate (HS) by means of a linker were disclosed in WO2009/098147. These conjugates show potent antiviral activity. The use ofHS was motivated by the presence of at least two different HSrecognition sites in gp120, namely the V3 variable loop and the siteinduced by CD4 (CD4i): the CD4 moiety of the mCD4-HS is thought totrigger conformational changes in gp120 by direct interaction, thusresulting in exposure of the CD4i epitope, with which the covalentlybound HS can then interact, thereby impairing HIV virus infection of X4and R5 cell lines.

This approach consisting in inhibiting viral attachment to cells istherapeutically advantageous, since it directly targets the virus andnot the cells themselves. It is therefore devoid of the cellular effectsobserved with medication which binds to co-receptors. In addition, inview of the preservation of the sites involved as a function of variousviral tropisms, the compounds according to the invention should interactwith the gp120 of different viral isolates. While it might be misleadingto think that resistance will never arise, it can nevertheless beexpected that it should occur at a much lower rate than with othertreatments. Indeed, the CD4 site of gp120 has to remain intact in orderto continue to bind to CD4, like the basic residues involved in bindingto the polyanionic polysaccharide for interaction with the co-receptors.Mutation in any of these two sites should indeed result in a virus withreduced infectivity.

The present Inventors have now identified a new conjugated moleculecapable of blocking the entry of the HIV virus into the cells. They showthat the presence of heparan sulphate oligosaccharides in the mCD4conjugated molecule is not absolutely required for inhibiting the virusentry into X4 or R5 cell lines and that very effective antiviralactivity can also be obtained by replacing the previously used HSmolecule by anionic polypeptides consisting of 5 to 21, advantageously 5to 17, notably 5, 9, 13, 17 or 21, and preferably 13 amino acids. In apreferred embodiment, some amino acids are negatively charged, notablyat least 1, advantageously at least 2, and preferably at least 3. Inmore preferred embodiments, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 aminoacids are negatively charged (depending on the length of the anionicpolypeptide). In the most preferred embodiment 9 amino acids arenegatively charged when the anionic polypeptide consists of 13 aminoacids. More importantly, they have shown that, surprisingly, these newmCD4 conjugated molecules (comprising the said anionic polypeptides)have a better antiviral activity against both R5 or X4 viruses than theconjugated molecules disclosed in the prior art.

According to a first aspect, the invention covers a conjugated moleculecomprising a peptide derived from the CD4 receptor, said peptide beingcoupled to an organic molecule by means of a linker, wherein:

-   -   the said peptide derived from the CD4 receptor comprises the        following general sequence (I):

(I)Xaa^(f) - P1- Lys - Cys - P2 - Cys - P3 - Cys - Xaa^(g) - Xaa^(h) - Xaa^(i) - Xaa^(j) - Cys -Xaa^(k) - Cys - Xaa¹ - Xaa^(m),

in which:

-   -   P1 represents 3 to 6 amino acid residues,    -   P2 represents 2 to 4 amino acid residues,    -   P3 represents 6 to 10 amino acid residues,    -   Xaa^(f) represents N-acetylcysteine (Ac-Cys) or thiopropionic        acid (TPA),    -   Xaa^(g) represents Ala or Gln,    -   Xaa^(h) represents Gly or (D)Asp or Ser,    -   Xaa^(i) represents Ser or His or Asn,    -   Xaa^(j) represents biphenylalanine (Bip), phenylalanine or        [beta]-naphthylalanine,

Xaa^(k) represents Thr or Ala,

Xaa^(l) represents Gly, Val or Leu, and

Xaa^(m) represents —NH₂ or —OH,

the amino acid residues in P1, P2 and P3 being natural or non-natural,identical or different, said residues of P1, P2 and P3 being alldifferent from the Lys residue and P1, P2 and P3 having a sequence incommon or not, and

-   -   the said organic molecule comprises    -   an anionic polypeptide consisting of 5 to 21, advantageously 5        to 17, and preferably 13 amino acid residues being natural or        non-natural, identical or different, wherein at least 3, and        notably 3 to 15, such as 3 to 13 amino acids are negatively        charged,    -   a molecular group A-Z, wherein:        -   A comprises a group chosen between the groups of formula            —CO(CH₂)₃NH—CO(CH₂)₂—, —CO(CH₂)_(p)—NH—CO—(CH₂)_(q)—,            —CO(CH₂—CH₂)—(O—CH₂—CH₂)_(p)—NH—CO—(CH₂)_(q)—,            —CO(CH₂)_(p)—NH—CO—(CH₂—CH₂—O)_(q)—(CH₂—CH₂)— and            —CO(CH₂—CH₂)—(O—CH₂—CH₂)—NH—CO—(CH₂—CH₂—O)_(q)—(CH₂—CH₂)—,            wherein p represents an integer comprised between 1 and 10            and q represents an integer comprised between 1 and 10, and            wherein the first carbonyl group is coupled to the alpha NH₂            of N-terminal Serine, and advantageously A represents a            group of formula —CO(CH₂)₃NH—CO(CH₂)₂— and        -   Z represents an halogen atom, a thiol or a maleimide group,

the said anionic polypeptide being linked to the linker by the saidmolecular group of formula A-Z, and the said linker being covalentlybound at one of its extremity to the free amino group (—NH₂) of theamino acid residue Lys present in general sequence (I) of the saidpeptide derived from the CD4 receptor, and being covalently bound at itsother extremity to the Z group of the said organic molecule.

Preferably, P3 comprises at least one basic amino acid, said basic aminoacid being even more preferably arginine. The presence of basic residuesin this portion of the CD4 receptor fragment contributes to its bindingto the gp120 protein. The inventors therefore prefer to introduce atleast one basic amino acid into P3, preferably arginine. This maintainsthus a basic moiety which is not reactive at derivation at pH 7-8 butwhich has been found to be useful for the binding of miniCD4 peptide tothe gp120 protein.

In this application, the terms “miniCD4 peptide”, “CD4 peptide” and“miniCD4” are used interchangeably to designate the peptide derived fromthe CD4 receptor comprising or consisting of general sequence (I)defined above.

As disclosed in WO 2009/098147, it is required that the miniCD4 peptideof the invention contains one and only one lysine (Lys) amino acidresidue, and that said lysine is in the position as defined in generalsequence (I). The Cys residues in general sequence (I) allow theformation of three disulphide bridges needed for folding back ofminiCD4. Thiopropionic acid (TPA), when it is in the N-terminus positionof the peptide of general sequence (I), makes it possible to reducehindrance in N-ter and overcome the presence of an amine group. Thus,according to a preferred embodiment, Xaa^(f) represents TPA in generalsequence (I).

Bip increases contact with glycoprotein gp120 in the cavity where thePhe 43 of CD4 receptor is lodged. Thus, in a preferred embodiment,Xaa^(j) represents Bip. Nevertheless, it may be advantageous to have Pheas Xaa^(j) in general sequence (I), since a structural analysis suggeststhat such a miniCD4 may mimic CD4 efficiently (Huang C C et al.,Structure. 2005 May; 13(5):755-68). Thus according to another preferredembodiment, Xaa^(j) represents Phe.

The peptide of general sequence (I) derived from the CD4 receptor formsan alpha helix structure followed by a beta sheet. The amino acidsXaa^(g)-Xaa^(h)-Xaa^(i)-Xaa^(j)-Cys-Xaa^(k)-Cys-Xaa^(l) participate in amajor way to the binding to gp120. These peptides display an IC₅₀(affinity for gp120) similar to those of sCD4 (soluble CD4).

The CD4 peptide of the invention can be prepared by conventional solidphase chemical synthesis techniques, for example according to the Fmocsolid phase peptide synthesis method (“Fmoc solid phase peptidesynthesis, a practical approach”, edited by W. C. Chan and P. D. White,Oxford University Press, 2000) and/or by genetic recombination.

Preferably, the said CD4 peptide is chosen from the group consisting ofsequences SEQ ID No. 1 and SEQ ID No. 2. More preferably, the CD4peptide of the invention has the sequence represented by SEQ ID No. 1.

The term “linker” refers in the present invention to a linker obtainedby the coupling of a bifunctional compound, as defined below, with apeptide derived from the CD4 receptor and the organic molecule. Thus,the length of the linker varies as a function of the bifunctionalcompounds used.

In particular, the linker will be advantageously chosen among:

with k representing an integer comprised between 2 and 24, and beingadvantageously 2, 4, 8 or 12,

with k1 representing an integer comprised between 1 and 10, thus equalto 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and advantageously equal to 1, 2, 3,5 or 10,

when Z represents a thiol group, and among:

when Z represents a maleimide group or a halogen atom.

In a preferred embodiment, the linker will be chosen among

with k representing an integer comprised between 2 and 24, and beingadvantageously 2, 4, 8 or 12 and

with k1 representing an integer comprised between 1 and 10, thus equalto 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and advantageously equal to 1, 2, 3,5 or 10,

when Z represents a thiol group,

and among:

when Z represents a maleimide group or a halogen atom.

Such linkers correspond thus to the use ofsuccinimidyl-6-[beta-maleimidopropionamido]hexanoate (SMPH),NHS-PEO_(n)-maleimide, with n representing an integer comprised between2 and 24, and being advantageously 2, 4, 8 or 12, SATA(N-succinimidyl-S-acetylthioacetate) and SATP(N-succinimidyl-S-acetylthiopropionate), as bifunctional compound.

In another preferred embodiment, the linker is

The conjugated molecule of the invention comprises an anionicpolypeptide, said anionic peptide consisting of 13 amino acids, wherein3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 amino acid residues are negativelycharged. In a preferred embodiment, the anionic polypeptide consists of13 amino acids, wherein 9 amino acid residues are negatively charged.

Advantageously, the anionic peptide of the invention is linked to the Agroup of the compound of formula I by the N-terminal end.Advantageously, the bond formed between the said anionic peptide and theA group is a peptidic bond, and thus involves both the amino (NH₂) ofthe said peptide and the carboxylic group (COOH) on the A group.

Without being bound by theory, it is thought that the said 13 aminoacid-long anionic polypeptides impair the interaction between the CD4iexposed site and CXCR4 (or CCR5) chemokine receptors, thereby impairingthe initiation of the cell/virus membrane fusion.

In the sense of the present invention, “amino acids” means all naturalα-amino acid residues (for example alanine (Ala), arginine (Arg),asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamine (Gln),glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile),leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe),proline (Pro), serine (Ser), threonine (Thr), tryptophane (Trp),tyrosine (Tyr) and valine (Val)) in D or L form, as well as non-naturalamino acids (for example, β-alanine, allylglycine, tent-leucine,norleucine (Nle), 3-amino-adipic acid, 2-aminobenzoic acid,3-aminobenzoic acid, 4-aminobenzoic acid, 2-aminobutanoic acid,4-amino-1-carboxymethyl piperidine, 1-amino-1-cyclobutanecarboxylicacid, 4-aminocyclohexaneacetic acid, 1-amino-1-cyclohexanecarboxylicacid, (1R,2R)-2-aminocyclohexanecarboxylic acid,(1R,2S)-2-aminocyclohexanecarboxylic acid,(1S,2R)-2-aminocyclohexanecarboxylic acid,(1S,2S)-2-aminocyclohexanecarboxylic acid, 3-aminocyclohexanecarboxylicacid, 4-aminocyclohexanecarboxylic acid,(1R,2R)-2-aminocyclopentanecarboxylic acid,(1R,2S)-2-aminocyclopentanecarboxylic acid1-amino-1-cyclopentanecarboxylic acid, 1-amino-1-cyclopropanecarboxylicacid, 4-(2-aminoethoxy)-benzoic acid, 3-aminomethylbenzoic acid,4-aminomethylbenzoic acid, 2-aminobutanoic acid, 4-aminobutanoic acid,6-aminohexanoic acid, 1-aminoindane-1-carboxylic acid,4-aminomethyl-phenylacetic acid, 4-aminophenylacetic acid,3-amino-2-naphthoic acid, 4-aminophenylbutanoic acid,4-amino-5-(3-indolyl)-pentanoic acid, (4R,5 S)-4-amino-5-methylheptanoicacid, (R)-4-amino-5-methylhexanoic acid,(R)-4-amino-6-methylthiohexanoic acid, (S)-4-amino-pentanoic acid,(R)-4-amino-5-phenylpentanoic acid, 4-aminophenylpropionic acid,(R)-4-aminopimeric acid, (4R,5R)-4-amino-5-hyroxyhexanoic acid,(R)-4-amino-5-hydroxypentanoic acid,(R)-4-amino-5-(p-hydroxyphenyl)-pentanoic acid, 8-aminooctanoic acid,(2S,4R)-4-amino-pyrrolidine-2-carboxylic acid,(2S,4S)-4-amino-pyrrolidine-2-carboxylic acid, azetidine-2-carboxylicacid, (2S,4R)-4-benzyl-pyrrolidine-2-carboxylic acid,(S)-4,8-diaminooctanoic acid, tert-butylglycine, γ-carboxyglutamate,β-cyclohexylalanine, citruline, 2,3-diamino propionic acid, hippuricacid, homocyclohexylalanine, moleucine, homophenylalanine,4-hydroxyproline, indoline-2-carboxylic acid, isonipecotic acid,sulfotyrosine, aminosuberic acid, p-carboxymethyl phenylalanine,α-methyl-alanine, nicopetic acid, norvaline,octahydroindole-2-carboxylic acid, ornithine, penicillamine,phenylglycine (Phg), 4-phenyl-pyrrolidine-2-carboxylic acid, pipecolicacid, propargylglycine, 3-pyridinylalanine, 4-pyridinylalanine,1-pyrrolidine-3-carboxylic acid, sarcosine, the statins,tetrahydroisoquinoline-1-carboxylic acid,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, tranexamic acid,4,4-difluoro proline, 4-fluoro proline,alpha-(3,4-difluorobenzyl)-proline, gamma-(3,4-difluorobenzyl)-proline,alpha-(trifluoromethyl)phenylalanine, hexafluoroleucine,5,5,5-trifluoroleucine, 6,6,6-trifluoronorleucine,2-(trifluoromethyl)leucine, 2-(trifluoromethyl)norleucine,4,4,4-trifluorovaline, 4,4,4,4′,4′,4′-hexafluorovaline,pentafluorophenylalanine, 2,3-difluorophenylalanine,2,4-difluorophenylalanine, 2,5-difluorophenylalanine,2,6-difluorophenylalanine, 3,4-difluoropheny 1 alanine,3,5-difluorophenylalanine, 3,3-difluoro-3-(4-fluorophenyl)alanine,2,3-difluorophenylglycine, 2,4-difluorophenylglycine,2,5-difluorophenylglycine, 3,4-difluorophenylglycine,4,4-difluoroethylglycine, 4,4,4-trifluoroethylglycine andhexafluoronorleucine). The term also includes natural and non-naturalamino acids carrying a conventional amino protecting group (for example,an acetyl group, tert-butyloxycarbonyl, benzyloxycarbonyl or9-fluorenylmethylcarbonyl), as well as natural and non-natural aminoacids protected at the carboxylic end (advantageously by a C₁-C₁₈ alkylgroup, an ester, a phenyl amide or benzyl amide or an amide, which,respectively, give a carboxylic end of the following formula: —CO(C₁-C₁₈alkyl), —COO(C₁-C₁₈ alkyl), —CONHphenyl, CONHbenzyl, or CONH₂).

As used herein, the term “anionic” designates a negatively chargedmolecule, which for example migrates to an anode in electrolysis. Also,a “negatively charged” amino acid designates an amino acid which carriesa side-chain charge which is negative at a pH of 7.4. Negatively chargedamino acid residues according to the invention thus include, forexample, aspartic acid, tyrosine, sulfotyrosine, tyrosine sulfonate,aminosuberic acid, p-carboxymethyl phenylalanine, and glutamic acidresidues.

As used herein, sulfotyrosine (or2-amino-3-(4-(sulfooxy)phenyl)propanoic acid) is a non-natural aminoacid having the following formula:

As used herein, the term “tyrosine sulfonate” (or“2-amino-3-(4-(sulfomethyl)phenyl) propanoic acid”) refers to a nonnatural amino acid having the following formula:

According to the present invention, aminosuberic acid is a non-naturalamino acid having the following formula:

As used herein, p-carboxymethyl phenylalanine refers to a non-naturalamino acid having the following formula:

In one embodiment of the invention, the said anionic polypeptideconsists of 13 identical negatively charged amino acids. It can be, forexample, the polypeptide consisting of 13 aspartic acid residues (SEQ IDNO: 9), 13 sulfotyrosine residues (SEQ ID NO: 10), 13 tyrosine sulfonateresidues (SEQ ID NO: 11), 13 aminosuberic acid residues (SEQ ID NO: 12),13 p-carboxymethyl phenylalanine residues (SEQ ID NO: 13), and 13glutamic acid residues (SEQ ID NO: 3). In a preferred embodiment, saididentical amino acid is glutamic acid E and the anionic polypeptide ofthe conjugated molecule of the invention is SEQ ID NO: 3.

In another embodiment of the invention, the anionic peptide of theinvention comprises negatively charged and uncharged amino acids.Preferably, the said anionic peptide comprises at least one negativelycharged amino acid and at least another amino acid. Preferably, the saidother amino acid is an amino acid carrying a polar uncharged side chain.Such amino acids include e.g. serine, threonine, asparagine, andglutamine. In a further preferred embodiment, the said other amino acidis serine or threonine. In a still further preferred embodiment, thesaid other amino acid is serine. In another preferred embodiment, thenegatively charged amino acid is aspartic acid.

In another further preferred embodiment, the said anionic polypeptide ofthe invention comprises at least two different negatively charged aminoacids, in addition to the at least one other amino acid. In this case,said anionic polypeptide has for example the sequence S-(X-D-X-S)_(n),such as S-X-D-X-S-X-D-X-S-X-D-X-S, where n is an integer comprisedbetween 1 and 5, and preferably is 3, S represents serine, D representsaspartic acid, and X is selected from the group consisting of: tyrosine,sulfotyrosine, tyrosine sulfonate, aminosuberic acid, andp-carboxymethyl phenylalanine (SEQ ID NO: 19) and where the various Xgroups can be identical or different, preferably identical.

In this embodiment, said anionic polypeptide can have in particular anyof the following sequences:

-   -   S-(Y_(SO3)-D-Y_(SO3)-S)_(n), such as        S-Y_(SO3)-D-Y_(SO3)-S-Y_(SO3)-D-Y_(SO3)-S-Y_(SO3)-D-Y_(SO3)-S        (SEQ ID NO: 4), where Y_(SO3) is sulfotyrosine;    -   S-(Y_(SN)-D-Y_(SN)-S)_(n), such as        S-Y_(SN)-D-Y_(SN)-S-Y_(SN)-D-Y_(SN)-S-Y_(SN)-D-Y_(SN)-S (SEQ ID        NO: 5), where Y_(SO3) is tyrosine sulfonate;    -   S-(pF-D-pF-S)_(n), such as S-pF-D-pF-S-pF-D-pF-S-pF-D-pF-S (SEQ        ID NO: 6), where pF is p-carboxymethyl phenylalanine;    -   S-(Asu-D-Asu-S)_(n), such as        S-Asu-D-Asu-S-Asu-D-Asu-F-Asu-F-D-Asu-S (SEQ ID NO: 7), where        Asu is aminosuberic acid; and

S-(Y-D-Y-S)_(n), such as S-Y-D-Y-S-Y-D-Y-S-Y-D-Y-S (SEQ ID NO: 8), whereY is tyrosine.

The anionic polypeptide can have also the following sequences:

-   -   S-Y_(SO3)-D-Y_(SO3)-S-Y_(SO3)-D-Y-S-Y-D-Y-S (SEQ ID NO: 20), or    -   S-Y-D-Y-S-Y-D-Y_(SO3)-S-Y_(SO3)-D-Y_(SO3)-S (SEQ ID NO: 21).

Most preferably according to the invention, X represents sulfotyrosine.Therefore, in the most preferred embodiment, the anionic polypeptide ofthe invention has the sequence: S—(Y_(SO3)-D-Y_(SO3)-S)_(n), and inparticular S—Y_(SO3)-D-Y_(SO3)-S—Y_(SO3)-D-Y_(SO3)-S-Y_(SO3)-D-Y_(SO3)-S(SEQ ID NO: 4).

In a particular embodiment, the conjugated molecule of the inventioncomprises a peptide derived from the CD4 receptor, said peptide beingcoupled to an organic molecule by means of a linker, wherein:

-   -   the peptide derived from the CD4 receptor is chosen from the        group consisting of sequences SEQ ID NO: 1 and SEQ ID NO: 2,    -   the linker is CO—(CH₂O)₂CH₂NHCO(CH₂)₂-pyrrolidinyl-2,5-dione,        -   the organic molecule comprises an anionic polypeptide having            a sequence selected from the group consisting of:            S-(Y_(SO3)-D-Y_(SO3)-S)_(n),            S-(Y_(SN)-D-Y_(SN)-S)-S-(pF-D-pF-S)-S-(Asu-D-Asu-S)_(n), and            S-(Y-D-Y-S)_(n); and in particular selected from the group            consisting of:            S-Y_(SO3)-D-Y_(SO3)-S-Y_(SO3)-D-Y_(SO3)-S-Y_(SO3)-D-Y_(SO3)-S            (SEQ ID NO 4),            S-Y_(SN)-D-Y_(SN)-S-Y_(SN)-D-Y_(SN)-S-Y_(SN)-D-Y_(SN)-S (SEQ            ID NO: 5), S-pF-D-pF-S-pF-D-pF-S-pF-D-pF-S (SEQ ID NO: 6),            S-Asu-D-Asu-S-Asu-D-Asu-F-Asu-F-D-Asu-S (SEQ ID NO: 7),            S-Y-D-Y-S-Y-D-Y-S-Y-D-Y-S (SEQ ID NO: 8), said sequence            being linked to the linker by a molecular group of formula            A-Z, wherein A is —NHCO(CH₂)₃NH—CO(CH₂)₂— and Z is a thiol            group.

In a preferred embodiment, the conjugated molecule of the inventioncomprises the peptide derived from the CD4 receptor chosen from thegroup consisting of sequences SEQ ID No.1 and SEQ ID No.2, said peptidebeing coupled to an organic molecule by means of the linkerCO—(CH₂O)₂CH₂NHCO(CH₂)₂pyrrolidinyl-2,5-dione, and an anionicpolypeptide having the sequence S—(Y_(SO3)-D-Y_(SO3)-S)_(n), and inparticular S—Y_(SO3)-D-Y_(SO3)-S-Y_(SO3)-D-Y_(SO3)-S-Y_(SO3)-D-Y_(SO3)-S(SEQ ID NO 4), said sequence being linked to the linker by a moleculargroup of formula A-Z, wherein A is —NHCO(CH₂)₃NH—CO(CH₂)₂— and Z is athiol group.

The conjugates of the invention are capable of inhibiting HIV entry intothe cell, by blocking the attachment of the virus on the cell membraneand thereby the virus entry. The conjugates of the invention cantherefore be used in therapy. More preferably, the conjugates of theinvention can be used for treating viral infections. Even morepreferably, the said conjugates can be used for treating AIDS.Therefore, according to a second aspect, the invention covers aconjugated molecule as defined above for its use as medicament. Theinvention also relates to the conjugates as defined above for treatingviral infections, and, in particular, for the treatment of AIDS. The useof a conjugated molecule as defined above for the manufacture of amedicament for an antiviral treatment, and in particular for thetreatment of AIDS, is also an object of the present invention.

According to a third aspect, the invention covers a pharmaceuticalcomposition comprising a conjugated molecule as defined above and apharmaceutically acceptable vehicle. This composition can be used as amedicament, preferably for the treatment of viral infections, and, morepreferably, for the treatment of AIDS.

In the pharmaceutical compositions of the present invention for oral,intranasal, sublingual, subcutaneous, intramuscular, intravenous,transdermal, local or rectal administration, the active ingredient canbe administered in unit forms for administration, mixed withconventional pharmaceutical carriers, to animals or to humans. Suitableunit forms for administration comprise the forms for oraladministration, such as tablets, gelatin capsules, powders, granules andoral solutions or suspensions, the forms for sublingual and buccaladministration, the forms for subcutaneous, intramuscular, intravenous,intranasal or intraoccular administration and the forms for rectaladministration.

The pharmaceutical composition of the invention may contain, in additionto the carrier and conjugate of the invention, various diluents,fillers, salts, buffers, stabilizers, solubilizers, and other materialswell known in the art.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, buffers, salt solutions, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like that are physiologically compatible. The type ofcarrier can be selected based upon the intended route of administration.In various embodiments, the carrier is suitable for intravenous,intraperitoneal, subcutaneous, intramuscular, topical, transdermal ororal administration. Pharmaceutically acceptable carriers includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of media and agents for pharmaceutically activesubstances is well known in the art. A typical pharmaceuticalcomposition for intravenous infusion could be made up to contain 250 mlof sterile Ringer's solution, and 100 mg of the combination. Actualmethods for preparing parenterally administrable compounds will be knownor apparent to those skilled in the art and are described in more detailin for example, Remington's Pharmaceutical Science, 17th ed., MackPublishing Company, Easton, Pa. (1985), and the 18^(th) and 19^(th)editions thereof, which are incorporated herein by reference.

The conjugated molecule in the composition preferably is formulated inan effective amount. An “effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired result, such as induction of apoptosis in tumor cells. A“therapeutically effective amount” means an amount sufficient toinfluence the therapeutic course of a particular disease state. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the agent are outweighed by the therapeuticallybeneficial effects.

Dosage regimens may be adjusted to provide the optimum response. Forexample, a single bolus may be administered, several divided doses maybe administered over time, or the dose may be proportionally reduced orincreased. The compositions of the invention can be administered to asubject to affect viral infection in a subject. As used herein, the term“subject” is intended to include living organisms which is susceptibleto viral infection, and specifically includes mammals, such as rabbits,dogs, cats, mice, rats, monkey transgenic species thereof, andpreferably humans.

For therapeutic applications, the conjugated molecule of the inventionis administered to a mammal, preferably a human, in a pharmaceuticallyacceptable dosage form such as those discussed above, including thosethat may be administered to a human intravenously as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intracerebrospinal, subcutaneous, intraarticular,intrasynovial, intrathecal, oral, topical, or inhalation routes.

The invention also relates to an antiviral treatment method, preferablyan anti-AIDS treatment method, comprising the administration to apatient in need thereof of a conjugated molecule according to theinvention or the pharmaceutical composition containing it.

In a fourth aspect, the invention provides a process for the preparationof a conjugated molecule as defined above, characterized in that theprocess comprises the following steps:

-   -   a. contacting the miniCD4 peptide of general sequence (I) as        defined above with a bifunctional compound carrying two active        groups, so that one of the two active groups forms a covalent        bond with the free amino group (—NH₂) of the residue of the        amino acid Lys present in general sequence (I), in order to        obtain an activated peptide carrying the second active group of        the bifunctional group, and    -   b. contacting the activated peptide obtained at step (a) with an        organic molecule carrying a functional group as defined above        carrying a thiol group for which the thiol group (SH) has been        protected by a protective thiol group, so that the active group        of the activated peptide forms a covalent bond with the        functional group, protected or not, of the organic molecule, in        order to obtain the conjugated molecule.

The compound obtained at step (a) will be called indifferently, in thepresent application, “activated peptide”, “activated miniCD4”,“activated CD4 peptide” or “activated miniCD4 peptide”.

The functional group according to the invention refers to a halogenatom, a maleimide, a thiol or a protective thiol group.

The term “protective thiol group”, as used in the present inventionrefers to a sulfur atom substituted by a S-protecting group in order toprotect a thiol group against undesirable reactions during syntheticprocedures. Commonly used S-protecting groups are disclosed in Greene,“Protective Groups In Organic Synthesis” (John Wiley & Sons, New York(1981)). S-protecting groups comprise benzyl ethers, substituted or not,such as p-methoxybenzyl or p-nitrobenzyl, trityl ethers, thioethers,thioacetate or thioacetal. Advantageously, the protected thiol group isa thioacetyl.

When the organic compound carries a protective thiol group, saidprotective group will be deprotected before or during step (b), in orderto recover a free thiol group and to allow the coupling of this thiolwith the active group of the activated peptide.

The characteristics of the peptide derived from CD4 receptor are thesame as defined above. In particular, P3 comprises preferably at leastone basic amino acid, said basic amino acid being even more preferablyarginine. According to a preferred embodiment, Xaa^(f) represents TPA ingeneral sequence (I). According to another preferred embodiment, Xaa^(j)represents Phe. Preferably, the sequence of the peptide derived from theCD4 receptor of general sequence (I) is chosen from the group consistingof sequences SEQ ID NO: 1 and SEQ ID NO: 2, advantageously SEQ ID NO: 1.

The term “bifunctional compound” in this patent application refers toany compound incorporating two active groups wherein one of the twoactive groups is capable of forming a covalent bond with the free aminogroup (—NH₂) of the residue of the amino acid Lys present in generalsequence (I) and the other active group is capable of forming a covalentbond with the organic molecule.

The person skilled in the art knows well the bifunctional compoundswhich can be used within the framework of this invention. Namely, thebifunctional compound according to this invention can be chosen from thefollowing non-limiting list: NHS-PEO_(n)-Maleimide where n is comprisedbetween 2 and 24, advantageously n=2, 4, 8 or 12, Sulfo-KMUS(N-[k-maleimidoundecanoyloxy]sulfosuccinimide ester), LC-SMCC(succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxy-[6-amidocaproate]),KMUA (N-k-maleimidoundecanoic acid), SMPB (succinimidyl4[p-maleimidophenyl]butyrate), Sulfo-SMPB (sulfosuccinimidyl4[p-maleimidophenyl]butyrate), Sulfo-SIAB(N-sulfosuccinimidyl[4-iodoacetyl]aminobenzoate), SIAB (N-succinimidyl[4-iodoacetyl]aminobenzoate), Sulfo-EMCS([N-e-maleimidocaproyloxy]sulfosuccinimide ester), EMCA(N-e-maleimidocaproic acid), EMCS ([N-e-maleimidocaproyloxy]succinimideester), SMCC (succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate), Sulfo-SMCC(sulfosuccinimidyl[4N-maleimidomethyl]cyclohexane-1-carboxyl ate), MBS(m-maleimidobenzoyl-N-hydroxy succinimide ester), Sulfo-MBS(m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester), GMBS(N-[g-maleimidobutyryloxy]succinimide ester), Sulfo-GMBS(N-[g-maleimidobutyryloxy]sulfosuccinimide ester), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SBAP (succinimidyl3-[bromoacetamido]propionate), BMPS(N-[[beta]-maleimidopropyloxy]succinimide ester), BMPA(N-[beta]-maleimidopropionic acid), AMASN-(a-maleimidoacetoxy)succinimide ester), SIA (N-succinimidyliodoacetate), SMPH (succinimidyl-6-[beta-maleimidopropionamido]hexanoate), SATA (N-succinimidyl-5-acetylthioacetate) andSATP (N-succinimidyl-S-acetylthiopropionate).

According to the invention, NHS-PEO_(n)-Maleimide wherein n=2 is alsocalled succinimidyl-[(N-maleimidoproprionamido)-diethyleneglycol]ester,

-   NHS-PEO_(n)-Maleimide wherein n=4 is also called    succinimidyl-[(N-maleimidoproprionamido)-tetraethyleneglycol]ester,-   NHS-PEO_(n)-Maleimide wherein n=8 is also called    succinimidyl-[(N-maleimidoproprionamido)-octaethyleneglycol]ester,-   NHS-PEO_(n)-Maleimide wherein n=12 is also called    succinimidyl-[(N-maleimidoproprionamido)-dodecaethyleneglycol]ester.

The active group capable of forming a covalent bond with the free aminegroup (—NH₂) of the residue of amino acid Lys present in generalsequence (I) can be any active ester group.

Preferably, the active group capable of forming a covalent bond with thefree amine group (—NH₂) of the residue of amino acid Lys present ingeneral sequence (I) is the active group N-hydroxysuccinimide ester(NHS) or N-hydroxy-4-sulfo-succinimide ester, and advantageously is theNHS active group. Even more preferably, the two active groups of thebifunctional compound are different (heterobifunctional group) and oneof the two groups is the NHS active group or aN-hydroxy-4-sulfo-succinimide ester, and advantageously is the NHSactive group.

Advantageously, the active group of the bifunctional compound, capableof forming a covalent group with the functional group of the organicmolecule, is a halogen atom or a maleimide group when the functionalgroup of the organic molecule is a thiol or a protective thiol group andis a thiol or a protective thiol group, as defined above, when thefunctional group of the organic molecule is a halogen atom or amaleimide group.

According to a preferred embodiment, when the functional group of theorganic molecule is a thiol group or a protective thiol group, thebifunctional compound is chosen from the group consisting ofsuccinimidyl-6[beta-maleimidopropionamido]hexanoate (SMPH) andNHS-PEO_(n)-maleimide, n being comprised between 2 and 24, andadvantageously is 2, 4, 8 or 12.

According to a particularly preferred embodiment, the bifunctionalcompound is SMPH.

The molecular structure of SMPH is as follows:

According to yet another particularly preferred embodiment, thebifunctional compound issuccinimidyl-[(N-maleimidopropionamido)-diethyleneglycol]ester, alsocalled NHS-PEO₂-maleimide,succinimidyl-[(N-maleimidopropionamido)-tetraethyleneglycol]ester, alsocalled NHS-PEO₄-maleimide,succinimidyl-[(N-maleimidopropionamido)-octaethyleneglycol]ester, alsocalled NHS-PEO₈-maleimide,succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol]ester, alsocalled NHS-PEO₁₂maleimide, still more preferably the bifunctionalcompound is NHS-PEO₂-maleimide.

The molecular structure of NHS-PEO₂-maleimide is as follows:

According to another particularly preferred embodiment, when thefunctional group of the organic molecule is a halogen atom or amaleimide group, the bifunctional compound is chosen from the groupconsisting of N-succinimidyl-5-acetylthioacetate (SATA) andN-succinimidyl-5-acetylthiopropionate (SATP).

The molecular structure of SATA is as follows:

The molecular structure of SATP is as follows:

The bifunctional compounds can be obtained from PIERCE (Rockford, Ill.).

Preferably again, the process according to the invention includes apreliminary step for the preparation of the peptide derived from the CD4receptor of general sequence (I), when Xaa^(f) represents TPA, said stepconsisting of contacting the peptide derived from the CD4 receptor ofthe following general sequence (III):

(III)P1- Lys - Cys - P2 - Cys - P3 - Cys - Xaa^(g) - Xaa^(h) - Xaa^(i) - Xaa^(j) - Cys - Xaa^(k) - Cys -Xaa¹ - Xaa^(m), 

where P1 to P3 and Xaa^(g) to Xaa^(m) are as defined in general sequence(I), with N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) in orderto incorporate TPA at the N-terminus of said peptide derived from theCD4 receptor of general sequence (III).

The molecular structure of SPDP is as follows:

Moreover, as examples of active groups capable of coupling to an organicmolecule by means of a covalent bond, the following groups can be cited:maleimide or bromoacetyl, S—S-pyridinium of thioacetyl.

When miniCD4 is activated by a protected thiol group (e.g. thioacetyl),it is possible to carry out coupling to an organic molecule whichcarries a maleimide group for example. This is possible when thefunctionalisation of the polyanionic polypeptide by a thiol group orprotected thiol group, such as thioacetyl, poses a problem. This thencalled “reverse coupling”.

Preferably, the active group is the maleimide group.

The molecular structure of the activated peptide according to theinvention whose active group is maleimide is the following when SMPH isthe bifunctional compound used:

In this application, the term “SMPH activated miniCD4 peptide” refers toan activated peptide according to the invention whose amino acid Lysresidue is covalently bound, advantageously by an amine bond, to amaleimide active group via a linker derived from SMPH.

According to another advantageous embodiment, the molecular structure ofthe activated peptide according to the invention whose active group isthe maleimide group is the following when NHS-PEO₂-maleimide is thebifunctional compound used:

In this application, the term “maleimide activated miniCD4 peptide via aPEO₂ linker” refers to an activated peptide according to the inventionwhose amino acid Lys residue is covalently bound, advantageously by anamine bond, to a maleimide active group via a PEO₂ linker.

According to another preference, the active group is the thioacetylgroup.

For example, the molecular structure of the activated peptide accordingto this invention whose active group is the thioacetyl group is thefollowing when SATA is the bifunctional compound used:

Similarly, the molecular structure of the activated peptide according tothis invention whose active group is the thioacetyl group is thefollowing when SATP is the bifunctional compound used:

The thioacetyl group is a protected form of the thiol group. Todeprotect the thiol group, we use hydroxylamine for example. This stepis carried out simultaneously to coupling to the maleimide group carriedby the organic molecule.

In this application, the terms “SATA activated miniCD4 peptide” and“SATP activated miniCD4 peptide” refer to an activated peptide accordingto the invention whose amino acid Lys residue is covalently bound,advantageously by an amine bond, to a protected thiol group (e.g.thioacetyl) via a linker derived from SATA or SATP.

Thus, according to a particular embodiment, the active group of theactivated peptide is the maleimide group and the organic moleculecarries a thiol or thioacetyl group.

The molecular structure of a conjugated molecule according to theinvention, including a peptide derived from the CD4 receptor of generalsequence (I) coupled to a modified anionic polypeptide (polyanion)carrying a thiol or protected thiol group, such as a thioacetyl group,is as follows when SMPH was used as bifunctional compound for thecoupling:

It can also be the following conjugated molecule when NHS-PEO₂-maleimideis the bifunctional compound used:

According to another embodiment, the conjugated molecule according tothe invention comprises a peptide derived from the CD4 receptorcomprising or consisting of general sequence (I), preferably sequenceSEQ ID NO: 1, and an organic molecule carrying a maleimide or halogengroup.

According to another particular embodiment, the active group of theactivated peptide is the thioacetyl group and the organic moleculecarries a maleimide or halogen group.

For example, the molecular structure of such a conjugated moleculeincluding a peptide derived from the CD4 receptor of general sequence(I) coupled to an organic molecule carrying a maleimide group is asfollows when SATA is used for the coupling:

According to the invention, the anionic polypeptide (the organicmolecule of the present invention) can be prepared by any convenientsynthetic method known in the art. The operating conditions for theprocesses according to the invention for preparation of the activatedpeptide and conjugated molecule are well known to the person skilled inthe art as illustrated in the following examples.

The examples and figures below illustrate the invention but do not limitits scope in any way.

FIGURE LEGENDS

FIG. 1 discloses the structure of the peptides of the invention.

FIG. 2 represents the HPLC profile of the anionic polypeptide comprisedin the conjugated molecules of the invention (from up to down:P3Y_(SO3), P3Y, P3 pF, P3Asu, and E13).

FIG. 3 represents the HPLC profile of the anionic polypeptide of theinvention coupled to the S-acetylpropionate (SATP) group.

FIG. 4 represents the HPLC profile of the conjugated peptides of theinvention.

FIG. 5 represents the results of the binding assays performed by SurfacePlasmon Resonance (SPR—Biacore) techniques, and showing the interactionbetween mAb17b (15 μg/ml) and surfaces coated with gp120 from X4 (MN) orR5 (YU2) virus, pretreated with 100 nM of: mCD4 alone(A), mCD4-P3Y(B) ormCD4-P3YSO3(C). When surfaces are coated with mAb17b (D and G), theinteraction of gp120 from X4 (MN) or gp120 from R5 (YU2) depends on thepresence of mCD4 (which induce the exposition of mAb17b epitope), but isfully inhibited when mCD4-P3Y or mCD4-P3YSO3 were used instead of mCD4.When surfaces are coated with sCD4 (E and H) the interactions arepartially inhibited by mCD4 and fully inhibited by mCD4-P3Y ormCD4-P3YSO3. When surfaces are coated with heparin (F), the interactionis not inhibited by mCD4 and partially inhibited by mCD4-P3Y andmCD4-P3YSO3, the later being more active.

EXPERIMENTAL EXAMPLES Example 1 Synthesis of the Conjugated Molecules ofthe Invention 1.1. Synthesis of the peptides of formulaH-γ-Abu-SXDXSXDXSXDXS-OH with X═YSO3 (P3YSO3), Y (P3Y), Asu (P3Asu), orpF (P3 pF); H-γ-Abu-SY_(SO3)DY_(SO3)SY_(SO3)DYSYDYS-OH (Nter3Sulfates);H-γ-Abu-SYDYSYDY_(SO3) SY_(SO3)DY_(SO3)S-OH (Cter3Sulfates); andH-γ-Abu-(Glu)₁₃-OH (E13); where γ-Abu: NH₂—(CH₂)₃—CO

The following example describes the synthesis of modified peptides offormula H-γ-Abu-SXDXSXDXSXDXS-OH with X═Y_(SO3) (P3YSO3), Y (P3Y), Asu(P3Asu), or pF (P3 pF); H-γ-Abu-SY_(SO3)DY_(SO3) SY_(SO3)DYSYDYS-OH(Nter3 sulfates); H-γ-Abu-SYDYSYDY_(SO3)SY_(SO3)DY_(SO3)S—OH(Cter3sulfates); H-γ-Abu-(Glu)₁₃-OH (E13); where γ-Abu: NH₂—(CH₂)₃—CO.In this formula, the anionic peptide of the invention (except peptideE13) corresponds to SXDXSXDXSXDXS-OH (SEQ ID NO: 19), with X as above(identical or different).

The resulting modified peptides are:

P3YSO3 (SEQ ID NO: 14):NH2-(CH₂)₃-CO-S-Y_(SO3)-D-Y_(SO3)-S-Y_(SO3)-D-Y_(SO3)-S-Y_(SO3)-D-Y_(S03)-S P3Y (SEQ ID NO: 15): NH2-(CH₂)₃-CO-S-Y-D-Y-S-Y-D-Y-S-Y-D-Y-SP3pF (SEQ ID NO: 16):  NH₂-(CH₂)₃-CO-S-pF-D-pF-S-pF-D-pF-S-pF-D-pF-SP3 Asu (SEQ ID NO: 17): NH₂-(CH₂)₃-CO-S-Asu-D-Asu-S-Asu-D-Asu-F-Asu-F-D-Asu-S E13 (SEQ ID NO: 18):  NH₂-(CH₂)₃-CO-EEEEEEEEEEEEENter3sulfates (SEQ ID NO: 20): NH2-(CH₂)₃-CO-S-Y_(SO3)-D-Y_(SO3)-S-Y_(SO3)-D-Y-S-Y-D-Y-SCter3sulfates (SEQ ID NO: 21):NH2-(CH₂)₃-CO-S-Y-D-Y-S-Y-D-Y_(SO3)-S-Y_(SO3)-D-Y_(S03)-S

Reagents

Resins were purchased from RAPP Polymere GmbH. Fmoc AAs, HATU, NMP, DMF,TFA were from Applied Biosystems and Piperidin from Sigma. Fmoc-Tyr(SO3.NnBu4)-OH and Fmoc-γ-Aminobutyric-OH (γ-Abu) were from Novabiochem,(S)-Fmoc-2-amino-octanedioc acid-8-ter-butyl ester (Asu) fromPolypeptides and Fmoc-L-4 (O-tButylcarboxymethyl)-Phe-OH (pF) fromAnaspec. HPLC grade triethylamine acetate buffer was from GlenResearch.N-succinimidyl-5-acetylthiopropionate (SATP) was from Pierce.

General Protocol for Peptide Synthesis

Peptides were synthesized on H-Ser(tBu)-2-ClTrt-PS-resin (100 μmoles;0.78 mmole/g) on an Applied 433 peptide synthesizer. E13 peptide wassynthesized on Fmoc-Glu(tBu)-PHB-PS-resin (100 μmoles; 0.61 mmole/g).Chain elongation was performed using 10 equivalents of Fmoc amino acidsand HATU/DIEA activation. Peptides were released from the resin byTFA/TIS/H2O (95/2.5/2.5) treatment for 1 h 30 at room temperature,except for sulphated peptides which was done at 4° C. (ice bath). Thecrude peptides were isolated by cold diethyl ether precipitation,solubilised in water by adding 3% NH₄OH, except the sulphated peptidethat was rapidly dissolved in 100 mM ammonium hydrogen carbonate buffer.After lyophilisation, the crude peptides were purified by C18 RP-HPLCusing 50 mM aqueous NEt3-AcOH (100 mM for the sulphated and E13peptides) and CH₃CN as eluents. Purified peptides was checked by massspectrometry (Waters ionspray Q-TOF-micro) and quantified by amino acidsanalyses (Hitachi L-8800 apparatus). Peptides purity was controlled byanalytical C18 RP-HPLC using linear gradient of CH₃CN in 50 mM aqueousNEt3-AcOH over 20 min (Waters Symmetry C18-300 Å, 3.5 2.1×100 mm column,0.35 ml/min flow rate).

TABLE 1 analysis of peptide purity by HPLC Overall HPLC yield Expectedmass retention time Peptide (%) Formula (monoisotopic) Found (min)P3YSO3 4 C₈₂H₉₈N₁₄O₄₉S₆ 2253.3835 [M − H]⁻ 2253.3164 8.3 (10-30% over 20min) P3Y 8 C₈₂H₉₈N₁₄O₃₁ 1775.6601 [M + H]⁺ 1775.6370 11.7 P3pF 14C₉₄H₁₁₀N₁₄O₃₇ 2027.7235 [M + H]⁺ 2027.7490 6.2 P3Asu 10 C₇₆H₁₂₂N₁₄O₃₇1823.8174 [M + H]⁺ 1823.8474 16.5 (0-10% over 20 min) E13 73C₆₉H₁₀₀N₁₄O₄₁ 1781.6170 [M + H]⁺ 1781.6299 12.1 Nter3 41 C₈₂H₉₈N₁₄O₄₀S₃2015.9495 2015.6544 12.242 (5-25% sulfates M (average) over 20 min)Cter3 46 C₈₂H₉₈N₁₄O₄₀S₃ 2015.9495 2015.6346 12.454 (5-25% sulfates M(average) over 20 min)

1.2. S-Acetylthiopropionate Peptides (SATP Peptides)

The peptides were dissolved in 100 mM sodium phosphate buffer pH 8.2 (1mM final concentration). S-acetylthiopropionate group was introduced viastepwise addition of 10 equivalents of SATP (0.26 M in DMSO) over a 40min period. After 1 h30, S-acetylthiopropionate peptides were purifiedby C18 RP-HPLC using linear gradient of CH₃CN in 50 mM aqueous NEt₃-AcOHover 20 min (C18-300 Å, 5 μm, 10×250 mm column, 6 ml/min flow rate).SATP derived peptides purity was controlled by analytical C18 RP-HPLCusing linear gradient of CH₃CN in 50 mM aqueous NEt3-AcOH over 20 min(Waters Symmetry C18-300 Å, 3.5 μm, 2.1×100 mm column, 0.35 ml/min flowrate).

TABLE 2 analysis of the purity of the S-acetylthiopropionate peptidesHPLC retention time (min) Peptide- Yield Expected mass (5-25% over SATP(%) Formula (monoisotopic) Found 20 min) P3YSO3- 60 C₈₇H₁₀₄N₁₄O₅₁S₇2383.3942 [M − H]⁻ 2383.4316 11.6 SATP P3Y- 43 C₈₇H₁₀₄N₁₄O₃₃S₁ 1903.6533[M − H]⁻ 1903.6781 16.0 SATP P3pF- 50 C₉₉H₁₁₆N₁₄O₃₉S₁ 2155.7167 [M − H]⁻2155.7869 9.3 SATP P3Asu- 36 C₈₁H₁₂₈N₁₄O₃₉S₁ 1953.8262 [M + H]⁺1953.7822 15.2* SATP E13- 34 C₇₄H₁₀₆N₁₄O₄₃S₁ 1909.6181 [M − H]⁻1909.6188 6.3 SATP Nter3 52 C₈₇H₁₀₄N₁₄O₄₂S₄ 2146.1159 2146.1746 11.0(10-30% sulfates- M (average) over 20 min) SATP Cter3 57 C₈₇H₁₀₄N₁₄O₄₂S₄2146.1159 2146.1357 11.3 (10-30% sulfates- M (average) over 20 min) SATP*10-30% linear gradient of CH₃CN in 0.08% aqueous TFA over 20 min.

1.3. Maleimide Activated miniCD4 (mCD4-Mal)

The mini-CD4 peptide of the invention is obtained as disclosed in WO2009/098147 and Nature Chemical Biology, 2009, 5(10), 743-748.

Mini-CD4-PEO2-maleimide=mini-CD4+linker

A solution of 10 mg of mCD4 (MW: 2897; 3.4 mmoles) in 1 ml of H₂O wasdiluted in 1 ml of phosphate buffer 0.1 M pH 8. 4.5 mg ofNHS-PEO₂-Maleimide (MW: 325; 13.8 mmoles; 4 equiv) were added to thiscloudy solution in 20 μl of DMSO with stirring. After 10 minutes, 85%(HPLC) of the starting materials was converted into maleimidederivative. Because of the low stability of the maleimide group at pH 8,the coupling reaction was directly loaded onto a SepaK C18 columncalibrated with 10% CH₃CN in aqueous TFA 0.08%. The maleimide derivativewas eluted with 50% CH₃CN. After freeze drying, the compound was thenpurified on a semi preparative column.

1.4. mCD4-Peptide Conjugates

SATP Peptides were dissolved in 100 mM sodium phosphate buffer pH 7.2 (1mM final concentration). 100 equivalents of 0.5 M NH₂OH, HCl in 100 mMsodium phosphate buffer (pH adjusted to 7.2 by 4N NaOH) was added.Deprotection of the thiol function was monitored by HPLC. After 30 min,0.3 equivalent of mCD4-Mal in H₂O (1.5 mM) was added. After 30 min,mCD4-peptide conjugates were purified by C18 RP-HPLC using lineargradient of CH₃CN in 50 mM aqueous NEt3-AcOH over 20 min (C18-300 Å, 5μm, 10×250 mm column, 6 ml/min flow rate). mCD4-peptide conjugates werecontrolled by analytical C18 RP-HPLC using linear gradient of CH₃CN in50 mM aqueous NEt3-AcOH over 20 min (Waters Symmetry C18-300 Å, 3.5 μm,2.1×100 mm column, 0.35 ml/min flow rate), negative mode massspectrometry and quantified by amino acids analysis.

TABLE 3 HPLC analysis of the mCD4-peptide conjugates Expected HPLCretention Yield mass time (min) (20-40% Conjugates (%) Formula (average)Found over 20 min) mCD4- 47 C₂₂₁H₃₁₄N₅₄O₈₈S₁₃ 5552.0933 5551.5127 11.5P3YSO3 mCD4-P3Y 38 C₂₂₁H₃₁₄N₅₄O₇₀S₇ 5071.7081 5071.5005 12.8 mCD4- 67C₂₃₃H₃₂₆N₅₄O₇₆S₇ 5323.9318 5323.5850 10.7 P3pF mCD4- 23 C₂₁₅H₃₃₈N₅₄O₇₆S₇5119.8291 5119.5283 10.8 P3Asu mCD4-E13 58 C₂₀₈H₃₁₆N₅₄O₈₀S₇ 5077.57505077.1021 11.4 mCD4- 36 C₂₂₁H₃₁₄N₅₄O₇₉S₁₀ 5311.9007 5311.5166 13.9(20-35% over Nter3 20 min) sulfates mCD4- 43 C₂₂₁H₃₁₄N₅₄O₇₉S₁₀ 5311.90075311.4155 14.0 (20-35% over Cter3 20 min) sulfates

Synthesis of the control peptide mCD4-HS12 was performed as described inWO/2009/098147 and Nature Chemical Biology, 2009, 5(10), 743-748.

Example 2 Biacore Evaluation 2.1. Protocol

Two different gp120 were used, originating from a type X4 isolate (gp120MN) and a type R5 isolate (gp120 YU2). The capacity of differentmolecules to inhibit gp120 interactions with various receptors orco-receptors is measured by surface plasmonic resonance (Biacore).

To do this, proteins (in this case gp120 envelop proteins) can beimmobilized on the surface of a biosensor (Sensorchip CM 4 Biacore) inaccordance with the described procedure (Vives et al. J. Biol. Chem.279, 54327-54333, 2005). When injected on a surface coated with MN(X4)or YU2(R5) gp120 envelops, mCD4 binds and unmasks the CD4i epitope ongp120, responsible to envelop binding of mAb17b (FIG. 5A) (mAb17bantibody recognizes the epitope induced by CD4 and mimics co-receptorCCR5 or CXCR4). When mCD4 or the compound to be tested is injected ontothese surfaces, the interaction signal between gp120 and mAb17b ismeasured as a function of time (FIGS. 5 A to C).

In another set of experiments, mAb17b are coated on the surface, andgp120 alone (grey line) or pre-incubated with mCD4, or the compound tobe tested are injected onto these surfaces (FIGS. 5D and 5G). Theinteraction signal between gp120 and mAb17b is measured as a function oftime.

In another set of experiments, sCD4 is coated on the surface, and gp120alone (grey line) or pre-incubated with the compounds to be tested areinjected onto these surfaces (FIGS. 5E and 5H). The interaction signalbetween gp120 and sCD4 is measured as a function of time.

In another set of experiments, heparin is coated on the surface, andgp120 alone (grey line) or pre-incubated with the compounds to be testedare injected onto these surfaces (FIG. 5F). The interaction signalbetween gp120 and heparin is measured as a function of time.

The difference between the light line and the heavy lines shows theinhibitory capacity of the tested compound vis-à-vis interactionsbetween gp120 and the molecule immobilized on the biosensor.

2.2. Results

mCD4-P3Y_(SO3) Fully Inhibits mAb17b Binding on Gp120/mCD4 Complex.

When injected on gp120 surface coated with MN(X4) or YU2(R5) envelops,mCD4 binds and unmasks the CD4i epitope on gp120, responsible to envelopbinding of mAb17b (FIG. 5A). The same experiments performed withmCD4-P3Y and mCD4-P3Y_(SO3) (FIGS. 5B and 5C respectively) show thatmCD4-P3Y partially inhibits mAb17b binding to MN(X4) (FIG. 5B), whereasmCD4-P3Y_(SO3) fully inhibits mAb17b on MN(X4) and YU2(R5) (FIG. 5C).

mCD4-P3Y_(SO3) Fully Inhibits MN(X4) Envelop Binding on mAb17b and CD4and Partially on Heparin.

When injected on mAb 17b surface, MN envelop does not bind to 17bsurface (CD4i epitope is masked, light line, FIG. 5D). This bindingoccurs when gp120 envelop is in complex with mCD4. When injected incomplex with mCD4-P3Y_(SO3), the binding is fully inhibited (FIG. 5D).MN envelop binds to CD4 surface. When injected in complex withmCD4-P3Y_(SO3), the binding is fully inhibited (FIG. 5E), whereas MNbinding to heparin is only partially inhibited by mCD4-P3Y_(SO3) (FIG.5F).

mCD4-P3Y_(SO3) Fully Inhibits YU2(R5) Envelop Binding on mAb17b and CD4.

When injected on mAb17b surface, YU2 envelop partially binds to 17bsurface. This binding is enhanced when mCD4/YU2 complex is injected.This binding is fully inhibited by mCD4-P3Y_(SO3) (FIG. 5G). YU2 envelopbinds to CD4 surface. When injected in complex with mCD4-P3Y_(SO3), thebinding is fully inhibited (FIG. 5H). As YU2 envelop does not bind toheparin surface, the experiment on heparin surface was not performed.

Example 3 Antiviral Activity of the Peptides of the Invention on theX4-Tropic HIV-1-LAI and R5-Tropic HIV-1/Ba-L Strains 3.1. Protocol

The antiviral experiment have been performed as described inWO/2009/098147 and Nature Chemical Biology, 2009, 5(10), 743-748.

Briefly, the X4-tropic HIV-1-LAI (Barre-Sinoussi, Science 220, 868-71,1983) or the R5-tropic HIV-1/Ba-L (Gartner et al, Science 233, 215-9,1986) strains were amplified and titrated in vitro onPhytohemaglutinin-P (PHA-P)-activated peripheral blood mononuclear cells(PBMC). Tissue culture infectious doses were calculated using Kärber'sformula (Kärber, Arch. Exp. Path. Pharmak. 162, 480-483, 1931). For theantiviral assay, PHA-P-activated PBMC were pre-treated for 30 minuteswith six concentrations of each drug (1:5 dilutions between 0.5 μM and160 pM) and infected with one hundred 50% tissue culture infectiousdoses (TCID50) of either the X4-tropic LAI or R5-tropic Ba-L strain.Drugs were maintained throughout the culture, and cell supernatants werecollected at day 7 post-infection and stored at −20° C. Azidothymidine(AZT) was used in these experiments as an internal control. Viralreplication was measured by quantifying reverse transcriptase (RT)activity in cell culture supernatants using the RetroSys HIV RT kit(Innovagen). In parallel, cytotoxicity of the samples was evaluated inuninfected PHA-P-activated PBMC by a methyltetrazolium salt (MTT) assayon day 7. Experiments were performed in triplicate and 50, 70 and 90%effective doses and cytotoxic doses were calculated using SoftMaxProsoftware.

PHA-P-activated PBMC were treated with each of the drug underinvestigation (1:5 dilutions between 0.5 μM and 160 μM) and infectedwith 100 TCID₅₀ of either HIV-1-LAI (X4 tropic) or /Ba-L (R5 tropic)strain. Molecules and viruses were maintained throughout the culture,and cell supernatants were collected at day 7 post-infection from whichreverse transcriptase activity was quantified. Experiments wereperformed in triplicate and 50, 70 and 90% effective doses (ED), in nM(±S.D.) were calculated using SoftMaxPro software. None of thesemolecules showed cytotoxicity up to 1 μM.

3.2. Results

When used alone, none of the anionic peptides demonstrated antiviralactivity at the highest concentration tested (500 nM). However, whenconjugated to mCD4, they displayed inhibitory activity against the LAIand/or Ba-L strain, with effective doses giving 50% inhibition (EDO aslow as 0.5 nM for mCD4-P3YSO₃, which compares well to 1.4 nM formCD4-HS₁₂ (see table 4 below).

TABLE 4 anti-viral activity of the conjugated peptides of the invention(effective dose (ED, mean of triplicate determination), in nM (± s.d.),required to inhibit 50, 70 and 90% of HIV-1 replication) VIH-1-LAI(X4)VIH-1/Ba-L(R5) Average ±S.D* Average ±S.D P3YSO3 ED50 >500 — >500 —ED70 >500 — >500 — ED90 >500 — >500 — P3Y ED50 >500 — >500 — ED70 >500— >500 — ED90 >500 — >500 — P3pF ED50 >500 — >500 — ED70 >500 — >500 —ED90 >500 — >500 — P3Asu ED50 >500 — >500 — ED70 >500 — >500 — ED90 >500— >500 — E13 ED50 >500 — >500 — ED70 >500 — >500 — ED90 >500 — >500 —mCD4 ED50 201 161 >500 — ED70 242 169 >500 — ED90 317 187 >500 —mCD4P3YSO3 ED50 0.5 0.2 1.3 1.1 ED70 0.6 0.3 3.1 2.7 ED90 1.1 0.8 20 9.2mCD4 P3Y ED50 98 36 454 104 ED70 124 74 498 11 ED90 160 125 >500 — mCD4P3pF ED50 8.2 6.5 245 196 ED70 11 7.4 >500 — ED90 20 11 >500 — mCD4P3AsuED50 15 5 499 4.3 ED70 20 7 >500 — ED90 31 14 >500 — mCD4 E13 ED50 30 25435 159 ED70 36 31 451 121 ED90 48 41 >500 mCD4-PEO2- ED50 1.4 1.2 189.6 HP12L ED70 1.7 1.6 215 231 ED90 3.5 5.3 >500 — AZT ED50 11 6 8.7 7.0ED70 21 15 16 11 ED90 60 53 38 28 *SD: standard deviation

None of these molecules showed cytotoxicity up to the highest testeddose (0.5 μM).

Example 4 Antiviral Activity of mCD4-P3YSO₃ on the 92UG029, SF162,92US723, 96USHIPS4, 92HT599 and 98IN017 Strains 4.1. Protocol

The antiviral assay was extended to a series of more clinically relevantprimary strains, including 92UG029, SF162, 92US723, 96USHIPS4, 92HT599and 98IN017.

Phytohemagglutinin (PHA)-P-activated PBMCs were infected either with thereference lymphotropic HIV-1/LAI strain (Barre-Sinoussi, et al., 1983)or with the reference macrophage-tropic HIV-1/Ba-L strain (Gartner, etal., 1986). These viruses were amplified in vitro with PHA-P-activatedblood mononuclear cells. Viral stocks (including clinical isolates) weretitrated using PHA-P-activated PBMCs, and 50% tissue culture infectiousdoses (TCID50) were calculated using Kärber's formula (Kärber, 1931).Viruses (125 TCID50) were incubated for 30 min with five concentrations(1:5 dilutions between 500 nM and 320 pM) of each of the molecules to betested and added to 150 000 PBMCs (m.o.i.˜0.001). Cell supernatants werecollected at day 7 post-infection and stored at −20° C. In some cases,the compounds were added to the cells prior to viral challenge. Viralreplication was measured by quantifying reverse transcriptase (RT)activity in the cell culture supernatants using the Lenti RT ActivityKit (Cavisi) and AZT was used as reference anti-HIV-1 molecule. Inparallel, cytotoxicity was evaluated on day 7 in uninfectedPHA-P-activated PBMC using a colorimetric methyl-tetrazolium salt(MTS/PMS) assay (Promega). Experiments were performed in triplicate and50, 70 and 90% effective doses (ED) were calculated using SoftMaxProsoftware.

4.2. Results

As shown in Table 5 below, mCD4-P3YSO₃ displayed a high level ofantiviral activity, characterized by ED₅₀ in the range of 0.2 to 1.2 nMfor five of them, and 29 nM for HIV-1 98IN017, a Glade C virus. As forthe LAI and Ba-L strains, the mCD4 or P3YSO₃ were only poorly active orinactive, further supporting the very strong synergistic effect inducedby the coupling strategy. None of the molecules showed cytotoxicity atup to 1 μM.

TABLE 5 anti-HIV-1 activity of AZT, mCD4-P3YSO₃, P3YSO₃ and mCD4 againstclinical HIV-1 isolates (effective dose (ED, mean of triplicatedetermination), in nM (±s.d.), required to inhibit 50, 70 and 90% ofHIV-1 replication) Viral strain: 92UG029 SF162 92US723 96USHIPS4 92HT59998IN017 Clade-tropism A-X4 B-R5 B-R5/X4 B-R5/X4 B-X4 C-X4 AZT ED₅₀ 7 ± 08 ± 7   8 ± 0.1 19 ± 9  9 ± 4 8 ± 3 ED₇₀ 16 ± 3  13 ± 8  17 ± 1  27 ± 1122 ± 5  19 ± 5  ED₉₀ 61 ± 17 31 ± 3  59 ± 19 56 ± 15 110 ± 13  108 ± 25 mCD4- ED₅₀ 0.2 ± 0.0 0.3 ± 0.2 0.3 ± 0.1 1.2 ± 1   0.5 ± 0.2 29 ± 18P3YSO₃ ED₇₀ 0.3 ± 0.1 0.4 ± 0.3 0.35 ± 0.2  1.6 ± 1.2 1.3 ± 0.9 147 ± 9 ED₉₀ 0.8 ± 0.3 0.9 ± 0.2 0.45 ± 0.2    3 ± 1.4 3.5 ± 0.0 >500 P3YSO₃ED₅₀ >500 >500 >500 >500 >500 >500 ED₇₀ >500 >500 >500 >500 >500 >500ED₉₀ >500 >500 >500 >500 >500 >500 mCD4 ED₅₀ 403 ± 76  245 ± 155 23 ±1  >500 355 ± 155 >500 ED₇₀ >500 352 ± 105 34 ± 10 >500 >500 >500ED₉₀ >500 >500 52 ± 22 >500 >500 >500

We also observed that mCD4-P3YSO3 does not need to be preincubated withthe virus to be active. Indeed, addition of the molecule either to thecells, prior to the viral challenge or to the virus prior to the cellinfection return, identical results (cf. table 7).

TABLE 7 Anti-HIV-1 activity of AZT, mCD4-P3YSO3, P3YSO3 and mCD4 againstLAI HIV-1 (effective dose (ED, mean of triplicate determinations), in nM(±s.d.) required to inhibit 50, 70 and 90% of HIV-1 replication, whenthe compounds were preincubated either with the cells or with theviruses) Pre-treated cells Pre-treated viruses AZT ED₅₀ 16.5 ± 12   20 ±12 ED₇₀ 33 ± 18 38 ± 18 ED₉₀ 96 ± 11 111 ± 40  mCD4-P3YSO₃ ED₅₀ 0.5 ±0.2 0.5 ± 0.3 ED₇₀ 0.6 ± 0.2 0.7 ± 0.3 ED₉₀   1 ± 0.2 0.9 ± 0.2 P3YSO₃ED₅₀ >500 >500 ED₇₀ >500 >500 ED₉₀ >500 >500 mCD4 ED₅₀ 310 ± 190 406 ±94  ED₇₀ >500 474 ± 27  ED₉₀ >500 >500

Example 5 Antiviral Activity of mCD4-P3YSO₃ in Cellulo on SHIVsf162p3Cells 5.1. Protocol

TZN-bl cells that express Luceriferase under the control of the HIV LTRwere used. These cells express high levels of CD4 and CCR5 and can beeasily infected with HIV-1/-2 and SIV. Upon infection of these cells,viral Tat will induce transcription of Luciferase. The Luc signal isproportional to the amount of infection. We add a serial dilution of thecompound to be tested (mCD4, mCD4-HS12 or mCD4-P3YSO₃) to cells andsubsequently add virus. The obtained Luc signal is read out after 48 h.

The test was carried out in duplicate.

5.2. Results

The results obtained are presented in the table 6 below.

EC50 (nM) 1^(st) experiment 2^(nd) experiment mCD4 4617 6126 mCD4-HS12781 631 mCD4-P3YSO₃ 34 37

CONCLUSIONS

This study thus shows that relatively small synthetic molecules,comprising a 3 kDa CD4 mimetic linked to an anionic polypeptide canefficiently mimic several large gp120 ligands, including CD4 andcoreceptor binding site recognizing mAbs. Remarkably, the conjugatesdescribed in the present invention neutralize both R5- and X4-tropicHIV-1, a significant advantage since the efficacy of CCR5-specificantagonists could be jeopardized by the emergence of viral strains thatutilize CXCR4, for which no inhibitors are yet available.

Surprisingly, the peptides of the invention displayed a better antiviralactivity than the molecules of the prior art (mCD4-HS12) both on X4 andR5 virus strains and can thus enhance the inhibition of the HIV virusentry into host cells.

The conjugated molecule mCD4-P3Y_(SO3) has far better inhibitory effectthan any other tested compounds (ED50 of 0.5 and 1.3 againstrespectively X4 and R5).

ABBREVIATIONS

Fmoc: 9-fluorenylmethyloxycarbonyl

DMF: Dimethylformamide

HATU: hexafluorophosphate N-oxide ofN[(dimethylamino)-1H-1,2,3-triazolo[4,5-b]pyridine-1-ylmethylene]-N-methylmethanaminium

DIEA: diisopropylethylamine

SPDP: N-succinimidyl-3(2-pyridyldithio)propionate

TFA: trifluoroacetic acid

EDT: ethanedithiol

TIS: triisopropylsilane

DTT: 1,4-dithiothreitol

MPLC: medium pressure liquid chromatography

ES⁺MS: electrospray mass spectrometry, positive mode

GSH: reduced glutathion

GSSG: oxidised glutathion

HPLC: high-performance liquid chromatography

RP-HPLC: reverse phase high-performance liquid chromatography

SMPH: succinimidyl-6[β-maleimidopropionamido]hexanoate

SATP: N-succinimidyl-5-acetylthioproprionate

RT: room temperature

Rt: retention time

Cbz: benzyloxycarbonyl

pMBn: p-methoxybenzyl

Bn: benzyl

Ac: acetyl

Me: methyl

Et: ethyl

eq: equivalent

HRMS: high resolution mass spectrum

ESI: electrospray ionisation

LC-ESI-TOF-MS: Liquid chromatography/electrospray ionizationTime-of-Flight mass spectrometry

LCMS: Liquid chromatography/Mass spectrometry

DMSO: Dimethylsulfoxide

1. Conjugated molecule comprising a peptide derived from the CD4receptor, said peptide being coupled to an organic molecule by means ofa linker, wherein: the said peptide derived from the CD4 receptorcomprises the following general sequence (I): (I)Xaa^(f) - P1- Lys - Cys - P2 - Cys - P3 - Cys - Xaa^(g) - Xaa^(h) - Xaa^(i) - Xaa^(j) - Cys -Xaa^(k) - Cys - Xaa¹ - Xaa^(m), 

in which: P1 represents 3 to 6 amino acid residues, P2 represents 2 to 4amino acid residues, P3 represents 6 to 10 amino acid residues, Xaa^(f)represents N-acetylcysteine (Ac-Cys) or thiopropionic acid (TPA),Xaa^(g) represents Ala or Gln, Xaa^(h) represents Gly or (D)Asp or Ser,Xaa^(i) represents Ser or His or Asn, Xaa^(j) represents biphenylalanine(Bip), phenylalanine or [beta]-naphthylalanine, Xaa^(k) represents Thror Ala, Xaa^(l) represents Gly, Val or Leu, and Xaa^(m) represents —NH₂or —OH, the amino acid residues in P1, P2 and P3 being natural ornon-natural, identical or different, said residues of P1, P2 and P3being all different from the Lys residue and P1, P2 and P3 having asequence in common or not, and the said organic molecule comprises ananionic polypeptide consisting of 5 to 21 amino acid residues beingnatural or non-natural, identical or different, wherein at least 3 aminoacids are negatively charged, a molecular group A-Z, wherein: Acomprises a group chosen between the groups of formula—CO(CH₂)₃NH—CO(CH₂)₂—, —CO(CH₂)_(p)—NH—CO—(CH₂)_(q),—CO(CH₂—CH₂)—(O—CH₂—CH₂)_(p)—NH—CO—(CH₂)_(q),—CO(CH₂)_(p)—NH—CO—(CH₂—CH₂—O)_(q)—(CH₂—CH₂)— and—CO(CH₂—CH₂)—(O—CH₂—CH₂)_(p)—NH—CO—(CH₂—CH₂—O)_(q)—(CH₂—CH₂)—, wherein prepresents an integer comprised between 1 and 10 and q represents aninteger comprised between 1 and 10, and wherein the first carbonyl groupis coupled to the alpha NH₂ of N-terminal serine, and advantageously Arepresents a group of formula —CO(CH₂)₃NH—CO(CH₂)₂—, and Z represents anhalogen atom, a thiol or a maleimide group, the said anionic polypeptidebeing linked to the linker by the said molecular group of formula A-Z,and the said linker being covalently bound at one of its extremity tothe free amino group (—NH₂) of the amino acid residue Lys present ingeneral sequence (I) of the said peptide derived from the CD4 receptor,and being covalently bound at its other extremity to the Z group of thesaid organic molecule.
 2. Conjugated molecule according to claim 1,wherein the sequence of the peptide derived from the CD4 receptor ofgeneral sequence (I) is chosen from the group consisting of sequencesSEQ ID NO: 1 and SEQ ID NO:
 2. 3. Conjugated molecule according to claim1 or 2, wherein the linker is chosen from the group consisting of:CO—(CH₂O)₂CH₂NHCO(CH₂)₂ pyrrolidinyl-2,5-dione

with k representing an integer comprised between 2 and 24,

with k1 representing an integer equal to 1, 2, 3, 5 and 10,

when Z represents a thiol group, and among:

when Z represents a maleimide group or a halogen atom.
 4. Conjugatedmolecule according to any one of claims 1 to 3, wherein the negativelycharged amino acids of the anionic polypeptide are chosen among asparticacid, tyrosine, sulfotyrosine, tyrosine sulfonate, aminosuberic acid,p-carboxymethyl phenylalanine, and glutamic acid, wherein saidsulfotyrosine has the formula:

wherein said tyrosine sulfonate has the formula:

wherein said aminosuberic acid has the formula:

and wherein said p-carboxymethyl phenylalanine has the formula:


5. Conjugated molecule according to any one of claims 1 to 4, whereinthe anionic polypeptide consists of 13 amino acids.
 6. Conjugatedmolecule according to any one of claims 1 to 5, wherein the anionicpolypeptide consists of identical amino acids.
 7. Conjugated moleculeaccording to claim 6, wherein said identical amino acid is glutamic acid(E).
 8. Conjugated molecule according to any one of claims 1 to 5,wherein the anionic polypeptide comprises at least two different aminoacids.
 9. Conjugated molecule according to claim 8, wherein the anionicpolypeptide comprises at least aspartic acid (D) and serine (S). 10.Conjugated molecule according to claims 8 and 9, wherein said anionicpolypeptide has a sequence of S-(X-D-X-S)_(n), such asS-X-D-X-S-X-D-X-S-X-D-X-S (SEQ ID NO: 19), where n represents an integercomprised between 1 and 5, S represents serine and D aspartic acid, andwhere X is selected from the group consisting of: tyrosine,sulfotyrosine, tyrosine sulfonate, aminosuberic acid, andp-carboxymethyl phenylalanine, and is preferably sulfotyrosine. 11.Conjugated molecule according to claim 10, wherein said anionicpolypeptide has a sequence which is selected in the group consisting of:S-(Y-D-Y-S)_(n), S-(Y_(SO3)-D-Y_(SO3)-S)_(n), S-(Y_(SN)-D-Y_(SN)-S)_(n),S-(pF-D-pF-S)_(n), and S-(Asu-D-Asu-S)_(n), where n represents aninteger comprised between 1 and 5, S represents serine, D representsaspartic acid, Y represents tyrosine, Y_(SO3) represents sulfotyrosine,Y_(SN) represents tyrosine sulfonate, pF represents p-carboxymethylphenylalanine and Asu represents aminosuberic acid.
 12. Conjugatedmolecule according to claim 11, wherein said anionic polypeptide has asequence which is selected in the group consisting of:S-Y-D-Y-S-Y-D-Y-S-Y-D-Y-S (SEQ ID NO: 8),S-Y_(SO3)-D-Y_(SO3)-S-Y_(SO3)-D-Y_(SO3)-S-Y_(SO3)-D-Y_(SO3)-S (SEQ IDNO: 4), 5-Y_(SN)-D-Y_(SN)-S-Y_(SN)-D-Y_(SN)-S-Y_(SN)-D-Y_(SN)-S (SEQ IDNO: 5), S-pF-D-pF-S-pF-D-pF-S-pF-D-pF-S (SEQ ID NO: 6),S-Asu-D-Asu-S-Asu-D-Asu-F-Asu-F-D-Asu-S (SEQ ID NO: 7),S-Y_(SO3)-D-Y_(SO3)-S-Y_(SO3)-D-Y-S-Y-D-Y-S (SEQ ID NO: 20), andS-Y-D-Y-S-Y-D-Y_(SO3)-S-Y_(SO3)-D-Y_(SO3)-S (SEQ ID NO: 21), where Srepresents serine, D represents aspartic acid, Y represents tyrosine,Y_(SO3) represents sulfotyrosine, Y_(SN) represents tyrosine sulfonate,pF represents p-carboxymethyl phenylalanine and Asu representsaminosuberic acid.
 13. Conjugated molecule according to any one ofclaims 1 to 12, wherein: the peptide derived from the CD4 receptor ischosen from the group consisting of sequences SEQ ID NO: 1 and SEQ IDNO: 2, the linker is CO—(CH₂O)₂CH₂NHCO(CH₂)₂pyrrolidinyl-2,5-dione, theorganic molecule comprises an anionic polypeptide having the followingsequence S-Y_(SO3)-D-Y_(SO3)-S-Y_(SO3)-D-Y_(SO3)-S-Y_(SO3)-D-Y_(SO3)-Sas defined in claim 12, which is linked to the linker by a moleculargroup of formula A-Z, wherein A is —NHCO(CH₂)₃NH—CO(CH₂)₂— and Z is athiol group.
 14. Conjugated molecule according to any one of claims 1 to13, for its use as medicament.
 15. Conjugated molecule according to anyone of claims 1 to 13, for its use for the treatment of AIDS. 16.Pharmaceutical composition comprising a conjugated molecule according toany one of claims 1 to 13 and a pharmaceutically acceptable vehicle. 17.Process for the preparation of a conjugated molecule according to anyone of claims 1 to 13, characterized in that the process comprises thefollowing steps: a. contacting the peptide derived from the CD4 receptorof general sequence (I) as defined in claim 1 with a bifunctionalcompound carrying two active groups, so that one of the two activegroups forms a covalent bond with the free amino group (—NH₂) of theresidue of the amino acid Lys present in general sequence (I), in orderto obtain an activated peptide carrying the second active group of thebifunctional group and b. contacting the activated peptide obtained atstep (a) with an organic molecule carrying a functional group as definedin claim 1 or with an organic molecule corresponding to the organicmolecule carrying a thiol group defined in claim 1 for which the thiolgroup (SH) has been protected by a protective thiol group, so that theactive group of the activated peptide forms a covalent bond with thefunctional group, protected or not, of the organic molecule, in order toobtain the conjugated molecule.